Substrate chromogen system

Paraffin-embedded 5-µm kidney sections were deparaffinized and rehydrated. Endogenous peroxidase activity was first quenched by H₂O₂ peroxidase blocking reagent (DakoCytomation). Macrophages and neutrophils were detected by using an anti-F4/80 antibody (1:50; eBioscience) and an anti–Ly-6G antibody (1:50; BD Biosciences), respectively; for 30 minutes at room temperature (RT). Sections were then washed and incubated with a secondary biotinylated goat anti-rabbit antibody (1:500; Jackson Immunoresearch) for 30 minutes at RT. Streptavidin-HRP was added and coloration was revealed using diaminobenzidine (DAB) with the substrate chromogen system from Dakocytomation. The number of F4/80⁺ and Ly-6G⁺ cells was counted in 10 non-overlapping fields (x400 magnification). Nitrotyrosine staining was performed using an anti-nitrotyrosine antibody (1:400; Abcam) and the OptiView DAB IHC Detection Kit (Ventana Medical Systems) according to manufacturer’s instructions. Nitrotyrosine intensity in the renal cortex was quantified using NIH ImageJ software.

Specimens were fixed in formalin and embedded in paraffin in accordance with the routine protocol implemented at the Pathology Department from CHUSJ. Sequential 5 μm sections, from the most representative tumor region and selected by a Pathologist, were stained with antibodies against CD68 (Dako, PG-M1), CD80 (R&D, MAB140), and CD163 (Novocastra, MRQ-26). Briefly, tissues were deparaffinized, hydrated and endogenous peroxidase activity was blocked with 3% methanol in hydrogen peroxide for 10 min. Following antigen retrieval in a water bath at 98°C with Tris EDTA, pH9 (CD68, 20 min) or citrate buffer, pH6 (CD80, 20 min; CD163, 40 min), primary antibodies were incubated as follows: CD80 overnight (1:50) at 4°C, CD68 30 min (1:100) and CD163 30 min (1:100), both at room temperature. After washing, labeled polymer secondary antibody (Envision Detection System, Dako) was added to slides and peroxidase activity was detected using diaminobenzidine (DAB) –tetrahydrochloride liquid plus substrate Chromogen System (Dako). The reaction was stopped with distilled water and sections were counterstained with haematoxylin and mounted in Richard-Allan Scientific Mounting Medium (ThermoFisher).

Formalin-fixed paraffin-embedded specimens were cut into 4 μm sections. After deparaffinization, sections were heated with an autoclave in Tris/HCl buffer (pH 9.0) for 20 min at room temperature for antigen retrieval. The sections were then incubated with 0.3% H₂O₂ in absolute methanol for 30 minutes and then incubated with primary antibody against caspase-3 (#PA1-29157, Thermo Fisher Scientific Co., USA) at concentration 1 : 1000. This was followed by sequential 60-minute incubations with secondary anti-rabbit antibody, Envision + System HRP-Labelled Polymer (Dako, USA), and visualization with liquid DAB (diaminobenzidine) substrate chromogen system (Dako, USA). All slides were lightly counterstained with hematoxylin for 30 seconds prior to dehydration and mounting [34 (link)].

Ear sections were deparaffinized and rehydrated using the Leica ST5020. Samples underwent heat-induced epitope retrieval, in sodium citrate, pH 6.0 at 95ºC for 20 minutes. The sections were then blocked for non-specific enzymatic reactions with Dual Endogenous Enzyme Block (cat. # S2003, Dako) for 10 minutes at room temperature. This was followed by blocking for non-specific protein interactions with Protein Block Serum-free (cat. # X0909, Dako) for 20 minutes at room temperature. Subsequent staining and detection with polyclonal rabbit anti-human CD3 (cat. # A0452, Dako) for 1 hour, followed by Dako EnVision+ system-HRP anti-rabbit polymer (cat. # K4002, Dako) for 30 minutes was performed. DAKO® Liquid DAB (3,3′diaminobenzidine tetrahydrochloride) + Substrate-Chromogen System (cat. # K3467, Dako) was used for 3 minutes, then counterstained for 1 minute with Gills hematoxylin for visualization of antigen-antibody reactions. Sections were then dehydrated on the Leica ST5020 Multistainer and coverslipped using a Leica CV5030. A USDA pathologist, who was blinded to the treatment groups, evaluated and scored the slides.

Microvessels in the rat hippocampus were colorized using the polyclonal rabbit anti-laminin antibody (dilution 1:1000; Dako, Glostrup, Denmark, No. Z009701) with negative immunohistochemistry controls. Laminin is a marker for basal lamina present in all microvessels, including capillaries, venules, and arterioles. The sections were deparaffinized in xylene, rehydrated and successively treated with cooled acetone for 10 min; Proteinase K for 6 min at room temperature; 5% normal goat serum for 20 min at room temperature; primary antibody solution for 36 h at 4°C; 50% N-Histofine Simple Stain MAX PO (Multi, Nichirei Biosciences Inc., Tokyo, Japan) for 30 min at room temperature; and, colorized for 1–4 min in liquid diaminobenzidine (DAB) Substrate Chromogen System (Dako, DAB Chromogen). Immunostained sections were counterstained with Gill’s hematoxylin. In the final step, sections were dehydrated in an alcohol series cleared by xylene, treated with mounting medium and coverslipped.

Selected sections were deparaffinized, rehydrated, and heated in a microwave oven in 0.01 M citrate buffer (pH 6.0; Química Contemporânea, Diadema, Brazil) for 30 min. Endogenous peroxidase activity was blocked by 3% hydrogen peroxide for 10 min, followed by a wash with phosphate buffered saline. The sections were incubated overnight at 4°C with the antibodies. The antibody was then detected using avidin-biotin peroxidase detection solution (Dakocytomation labelled streptavidin biotin reagent; Dakocytomation, Glostrop, Denmark and System-horseradishperoxidase; Dako, Glostrup, Denmark) and the signal was visualized using diaminobenzidine (Dakocytomation) and Substrate Chromogen-System (Dako). Slides were counterstained with Harris’s hematoxylin, dehydrated, cleared and mounted. Positive controls from the appendix and tonsils were used. The cells were initially observed at a low magnification (×100) to assess the general distribution of the antibody. The samples were subsequently examined at a higher magnification (×400). The evaluation of cell staining was performed in tumor tissue. The tumor cells (exhibiting gross and evident nucleoli, and irregular chromatin) were identified and counted at the higher magnification.

Selected liver sections were deparaffinized, rehydrated, and heated in a microwave oven in 0.01 M citrate buffer (pH 6.0; Química Contemporânea, Diadema, Brazil) for 30 min. Endogenous peroxidase activity was blocked by 3% hydrogen peroxide for 10 min, followed by a wash with PBS. Sections were incubated overnight at 4°C with the following primary antibodies: anti-FAS (1 : 50), anti-OX62 (1 : 10), and anti-TNFR1 (1 : 100). The primary antibody was then detected using avidin-biotin peroxidase detection solution (DakoCytomation labelled streptavidin biotin reagent; DakoCytomation, Glostrop, Denmark; and System-horseradishperoxidase; Dako, Glostrop, Denmark), and the signal was visualized using diaminobenzidine (DakoCytomation) and Substrate Chromogen System (Dako). Slides were counterstained with Harris's hematoxylin, dehydrated, cleared, and mounted. Positive controls from the appendix and tonsils were used. Cells were initially observed at a low magnification (×100) to assess the general distribution of the primary antibody. Samples were subsequently examined at a higher magnification (×400) [16 (link)]. Liver cells (exhibiting gross and evident nucleoli and irregular chromatin) were identified, and stained cells were counted at the higher magnification.